Genetic diversity and demographic history of the Old World Bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), in Ethiopia inferred from mitochondrial gene sequences

Abstract The Old World bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is a globally distributed agricultural and horticultural insect pest. Despite the economic importance of this insect in Ethiopia, its genetic diversity and demographic history are poorly understood. We examined the nucleotide variation of the mitochondrial cytochrome c oxidase subunit I (COI) gene fragment of 74 H. armigera individuals from six collection sites in Ethiopia. We recorded 15 COI haplotypes in H. armigera, ten globally shared and five exclusive to Ethiopia (HaET15, HaET14, HaET10, HaET7, and HaET4). Haplotype HaET1 was the most widely geographically distributed and frequent (71.62%). Analysis of molecular variance (AMOVA) revealed a high and significant level of variation within H. armigera populations (θST = −0.0135). Negative values of the neutrality test and nonsignificant index of mismatch distribution supported the demographic expansion of H. armigera populations in Ethiopia; furthermore, this was also supported by the nonsignificant values of the sum of squared deviations (SSD) and raggedness index (r). The high genetic variation and population expansion of H. armigera have immense implications for devising locally adapted management strategies in area‐wide integrated pest management IPM programs. However, a comprehensive study of H. armigera genetic diversity and population structure using various molecular markers is needed for future confirmation.


| INTRODUC TI ON
The Old World bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is a globally destructive insect pest (Fitt, 1989) that damages economically important crops (cotton, maize, pigeon pea, chickpea, tomato, beans, peas, sorghum, sunflower, niger seeds, etc.) (Sharma, 2005;Tebkew et al., 2002) across different ecological zones. Helicoverpa armigera has been reported as a key native insect pest in Ethiopia (Fite et al., 2018;Tebkew et al., 2002) and surrounding countries, such as Kenya (Kimurto et al., 2004), Tanzania (Maerere et al., 2010), and Sudan (Mansour & Mohmoud, 2014). The species has high reproductive and fecundity rates (Naseri et al., 2009;Razmjou et al., 2014) and is capable of extensive long-distance migration (Fitt, 1989) of up to 2000 km Jones et al., 2015) in a lifetime and up to 40 km in a single night (Jones et al., 2015). Up to two population peaks of H. armigera were reported in Ethiopia, beginning from June until the month of March, indicating that their population dynamics were dependent on various weather condition parameters (Fite et al., 2020). Furthermore, individuals of this species can tolerate a wide range of temperatures and drought by entering facultative diapause. Since 2013, the New World has been invaded by H. armigera (Czepak et al., 2013;Tay et al., 2013), which have Eurasian and African origins (Tay et al., 2017) and are typically confined to Africa, Europe, Asia, and Australasia (Sharma, 2005).
Such high mobility with explosive population growth, tolerance to varying temperatures and drought, and a polyphagous nature have probably been important factors contributing to the establishment of this species throughout most of the world. Wide geographical distributions, the availability of many alternative host plants (Brandvain et al., 2014;Peter & Slatkin, 2013) and climate change (Bonin et al., 2007;Willi et al., 2006) all contribute to the genetic variation in organisms.
Hypothetically, high within-population genetic variation and migration rates can provide the opportunity for new phenotypes or behaviors to emerge in pest populations (Zhou et al., 2000).
Additionally, ecological parameters (Peter Linder et al., 2013), the landscape, adaptations to climate change, and resistance to environmental change (Bonin et al., 2007;Willi et al., 2006) can also impose selection pressure on crop pests. Genetic studies provide useful information regarding the potential for large-scale insect pest control, particularly in species with extensive host ranges and wide geographical distributions (Alphey & Bonsall, 2018;Barman et al., 2012). Understanding the phylogenetic relationships among insect pest populations is critically important for informing effective and sustainable H. armigera management (Behere et al., 2007Tay et al., 2013). However, knowledge of the population genetic diversity and demographic history of H. armigera in Eastern Africa remains poor and geographically restricted.
Due to their relatively rapid evolutionary rates and haploid mode of maternal inheritance, with little or no recombination, mitochondrial DNA sequences can be used to infer recent femalespecific evolutionary histories (Avise et al., 1987). Studies make use of the mitochondrial cytochrome c oxidase subunit I (COI) gene to distinguish natural populations of lepidopterans that have adapted to different host plants (Ong'amo et al., 2008). COI genes have been effective in studies on phylogenetic relationships, genetic variables, demographic history and phylogeography in various insects (Ajao et al., 2021;Cao et al., 2019;Xu et al., 2019), for instance, Sesamia nonagrioides (Lepidoptera: Noctuidae) (Goftishu et al., 2019), Carposina sasakii (Lepidoptera: Carposinidae) (Wang et al., 2017), and H. armigera (Tay et al., 2017). We examined the genetic diversity and demographic history of the mitochondrial COI gene fragment of 74 individuals of H. armigera from six collection sites in Ethiopia.

| Sampling
Sampling was conducted in the Oromiya Regional State of central  (Table 1). A total of 74 H. armigera larvae were used for the analysis of genetic diversity and demographic history ( Table 1).
The collected larvae were kept in plastic vials for 24 h for starvation. Then, they were preserved in absolute ethanol and labeled with their host plants, site, date of collection, and GPS coordinates and stored at −20°C until required for DNA extraction.

| DNA extraction
Genomic DNA was extracted using the established protocols described by Behere et al. (2013) with modifications. Briefly, the absolute ethanol-preserved specimens were washed with sterilized distilled water and kept on a paper towel for 10 min at room temperature to allow the ethanol to evaporate and the insects to dry. Insect material consisting of the head and/or posterior end or whole larval instars estimated to weigh 100 mg was cut off with sterilized surgical blades. The cleaned larvae were ground in liquid nitrogen and genomic DNA was extracted using a Genomic II DNA Extraction Kit (BIOLINE) following the manufacturer's instructions. Genomic DNA was visualized on a 1% agarose gel for detection and an Eppendorf BioSpectrometer (Germany) to check the quality and quantity of the extraction protocols before being used for polymerase chain reaction (PCR).

| PCR amplification and sequencing
We amplified a 511-bp fragment of the COI gene by PCR in 10μl reaction volumes containing 6 μl of nucleus-free water, 2 μl of 5× HOT  Abbreviations: ChAD, populations collected from chickpea; CTK, populations collected from chili; NJR, populations collected from niger; PJR, populations collected from pea; SDD, populations collected from sunflower; TTK, populations collected from tomato.
initial denaturing at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min.
The amplified PCR products were sequenced by Macrogen Inc. after purification by an Exo 1-rSAP combination (Biolabs) according to the manufacturer's protocol. All the newly sequenced samples were deposited in the GenBank database of the NCBI. Tajima's D Tajima (Tajima, 1989a) and Fu's Fs (Fu, 1997) were estimated among populations. The spatial expansion hypothesis of

| Sequence analyses
Harpending's raggedness index (r) (Harpending et al., 1993) and the sum of squared deviations (SSD) were also tested using a parametric bootstrap approach with 1000 replicates using Arlequin (version indicated above). Similar software was used to test the hierarchical genetic structure of the populations based on analysis of molecular variance (amova).

| Genetic variation and diversity
We generated 402-bp mtDNA COI gene sequences of 74 H. armigera specimens obtained from the six populations across the sampling sites, all of which shared 99%-100% homology with the reference H. armigera sequences found in GenBank (https://www.ncbi.nlm. nih.gov/genba nk/) ( Table 1) Table 1).
For each of the population characteristics, polymorphisms were found in all six populations, which ranged from two for NJR to nine for PJR. The overall total polymorphic site number was 14. However, on a population basis, PJR had the most (9) polymorphic sites, followed by the ChAD and TTK populations ( which could be attributed to its low sample size, followed by PJR (  Madagascar, Chad, Cameroon, and Uganda), continents (Europe (Spain and France)), (South America (Brazil)), and the Caribbean region (Dominican Republic)). Generally, most of the haplotypes differed by 1-5 mutational steps from the central ancestor haplotype (Figure 2b).

| Population structure and demographic history
amova was performed to determine how the genetic variability was distributed among and within the populations (Table 4). amova did not suggest hierarchical genetic structure among the populations (Table 4). However, a high and significant percentage (101.35%) of the total variation occurred within populations (  (Lepidoptera: Noctuidae), which was reported from Africa, including

TA B L E 3 COI haplotype distribution in
Sudan (Hackett & Gatehouse, 1982); and H. peltigera (Schiffermuller) (Lepidoptera: Noctuidae), which is also present in most of Africa, such as Eritrea, Egypt, Sudan, Algeria, and Chad (Ahmed & Elamin, 1996), and the most economically important and widely distributed, H. armigera. Hence, the present investigation provides an efficient way to identify Helicoverpa species in the country. All sampled individuals from the six populations were confirmed to be H. armigera, representing the first molecular characterization of H. armigera in Ethiopia using the COI mtDNA gene region. DNA barcoding, such as using COI mtDNA, is an applicable and efficient method for the separation and confirmation of insect species (Foottit et al., 2008;Jung et al., 2010), including H. armigera (Leite et al., 2014).
Our data indicated lower haplotype diversity and higher nucleo-

| Haplotype distribution and network
We detected a total of 15 haplotypes in Ethiopian H. armigera populations using COI gene sequences. The majority of the haplotypes were randomly distributed throughout the six populations. The presence of unique haplotypes across the populations may be due to the small sample size used in this study. Leite et al. (2014)   .

| Population structure and demographic history
The weak population structure observed among the populations of H. armigera suggests that the variation was distributed randomly between populations due to the high gene flow resulting from migrations of the insect. Our results showed a lack of differentiation among the six studied populations of H. armigera, which was in line with reports from China, India, and Australia (Kraus et al., 2011;Scott et al., 2005;Weeks et al., 2010) and indicated that gene flow has been high enough to prevent trade-offs in fitness between H. armigera populations attacking diverse host plants from creating isolation. Based on several molecular markers, including allozymes, microsatellites and mtDNA, unstructured genetic networks of H. armigera distributed in other regions have been reported (Endersby et al., 2007;Nibouche et al., 1998). Therefore, the genetic variation observed in this study is not solely associated with differences in sampling sites. The biological characteristics allowed extensive movement of H. armigera with similar genetic make-ups/genetic homogeneity and allowed the insects to breed across the geographical populations we examined in Ethiopia. Compared to host-monophagous insects, polyphagous herbivorous insects such as H. armigera exhibit extensive plasticity in feeding depending on the host plant (Wang et al., 2017).
Demographic analysis using neutrality tests and a mismatch distribution analysis with COI mtDNA markers revealed expansion of H. armigera within the Ethiopian territory; a similar result was reported in Brazil by Leite et al. (2014) on the basis of similar molecular markers. When a genetic structure has been influenced by rapid range expansion, Tajima's D is expected to be negative, indicating an excess of rare nucleotide variants compared to the expected value under a neutral model of evolution Tajima (1989b). Similarly, Fu's F s test, which is based on the distribution of haplotypes, also showed negative values for H. armigera populations in Ethiopia, confirming an excess of rare haplotypes over the number that would be expected under neutrality (Fu, 1997). The nonsignificant values of SSD and r also support this interpretation. The observed nonsignificant values in goodness of fit distribution suggest that population expansion occurred recently (Rogers & Harpending, 1992) in populations of H. armigera in Ethiopia, which is also supported by the high within-population genetic variation and unique haplotypes observed in the present finding. A similar situation was previously found in insect species, with a high migration rate and from a small effective population size (Kraus et al., 2011), including insect pests such as Plutella xylostella, as inferred from COI mtDNA (Wei et al., 2013).   Data curation (equal); Methodology (equal); Supervision (equal);

ACK N OWLED G M ENTS
We gratefully acknowledge the research fund provided by USAID Moreover, we also acknowledge James Kabii and Peter Odhiambo for technical assistance, providing, and facilitating laboratory activities.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The sequence used in this study are deposited in the GenBank da-